Key words: hypoventilation syndrome; obesity; respiratory depression; subcutaneous fat; visceral fat

Fat Accumulation, Leptin, and Hypercapnia in Obstructive Sleep Apnea-Hypopnea Syndrome* Ryuhi Shimura, MD; Koichiro Tatsumi, MD, FCCP; Akira Nakamura, MD; Yasunori Kasahara, MD, FCCP; Nobuhiro Tanabe, MD, FCCP; Yuichi Takiguchi, MD, FCCP; and Takayuki Kuriyama, MD, FCCP Background: Obesity and visceral fat accumulation (VFA) are risk factors for the development of obstructive sleep apnea-hypopnea syndrome (OSAHS), and a subgroup of OSAHS patients acquire hypoventilation. Circulating leptin, an adipocyte-derived signaling factor, increases in accordance with body mass index (BMI); under experimental conditions, leptin selectively decreases visceral adiposity and it is also a respiratory stimulant. Objective: To investigate whether the location of body fat deposits, ie, the distribution of VFA and subcutaneous fat accumulation (SFA), contributes to hypoventilation and whether circulating levels of leptin are involved in the pathogenesis of hypoventilation, which is often observed in OSAHS. Methods: We assessed VFA and SFA by abdominal CT scan, and measured lung function and circulating levels of leptin in 106 eucapnic and 79 hypercapnic male patients with OSAHS. Results: In the whole study group, circulating leptin levels correlated with BMI (r 0.56), VFA (r 0.24), and SFA (r 0.47), but not with PO 2 or sleep mean arterial oxygen saturation (SaO 2 ). BMI, percentage of predicted vital capacity, FEV 1 /FVC ratio, apnea-hypopnea index, sleep mean SaO 2, VFA, and SFA were not significantly different between two groups. Circulating leptin levels were higher in the hypercapnic group than in the eucapnic group. Logistic regression analysis indicated that serum leptin was the only predictor for the presence of hypercapnia ( 0.21, p < 0.01). Conclusions: These results suggest that the location of body fat deposits may not contribute to the pathogenesis of hypoventilation, and circulating leptin may fail to maintain alveolar ventilation in hypercapnic patients with OSAHS. (CHEST 2005; 127:543 549) Key words: hypoventilation syndrome; obesity; respiratory depression; subcutaneous fat; visceral fat Abbreviations: AHI apnea-hypopnea index; BMI body mass index; CSF cerebrospinal fluid; OSAHS obstructive sleep apnea-hypopnea syndrome; Sao 2 oxygen saturation; SFA subcutaneous fat accumulation; VC vital capacity; VFA visceral fat accumulation *From the Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan. This study was supported by a Grant-in-Aid for Scientific Research (C)(14570541) from the Ministry of Education, Science, Sports and Culture, and grants to Respiratory Failure Research Group from the Ministry of Health, Labour and Welfare, Japan. Manuscript received February 26, 2004; revision accepted September 2, 2004. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions@chestnet.org). Correspondence to: Koichiro Tatsumi, MD, FCCP, Department of Respirology, Graduate School of Medicine, Chiba University, 1 8-1 Inohana, Chuou-ku, Chiba 260-8670, Japan; e-mail: tatsumi@ faculty.chiba-u.jp Leptin was first described as an adipose-derived hormone, which induces a complex response including control of body weight and energy expenditure after interaction with specific receptors located in the CNS and in peripheral tissues. 1 Leptin receptors are found in the hypothalamus, particularly in the arcuate nucleus, where leptin is thought to exert its primary feedback signaling. 2 Circulating levels of leptin reflect the amount of energy stored in adipose tissue and are reported to correlate with the body mass index (BMI) in humans. 3,4 Control of body weight is clinically important in patients with obstructive sleep apnea-hypopnea syndrome (OSAHS) because obesity, male gender, and increasing age are recognized to be risk factors for OSAHS. Among these risk factors, obesity plays a major role, because approximately 70% of patients with this disorder are obese and obesity is the only reversible risk factor of importance. 5 Among those with OSAHS, some individuals present an increase www.chestjournal.org CHEST / 127 / 2/ FEBRUARY, 2005 543

in resting Paco 2, leading to obesity hypoventilation syndrome. 6 Obesity itself is thought to affect the respiratory control system. The mechanical load imposed by obesity, especially visceral fat accumulation (VFA), on the respiratory system may explain the development of hypoventilation, although the majority of obese people breathe normally. 6 Alternatively, central defects of the respiratory control system may contribute to respiratory depression; however, the precise mechanisms have been undefined. 7 Leptin may be a modulator of the respiratory control system. The absence of leptin in the C57BL/ 6J-Lep ob mouse is associated with marked obesity, elevated Paco 2, and a reduced hypercapnic ventilatory response. 8 Conversely, leptin replacement in these mutant mice stimulated ventilation and hypercapnic ventilatory response across all sleep/wake states. The effects of leptin deficiency on respiratory depression, and the effects of leptin administration on respiratory control, were more pronounced during sleep than wakefulness in mice, although the precise mechanism by which leptin influences respiratory control has been undefined. 9,10 However, whether endogenous leptin plays a role in the respiratory control system in healthy humans and/or patients with OSAHS remains unclear. In addition, whether leptin affects visceral adiposity has not been determined in OSAHS, although it has been reported that leptin selectively decreases visceral adiposity in rats. 11 The purpose of the present study was to examine whether the location of body fat deposits, ie, the distribution of VFA and subcutaneous fat accumulation (SFA), contributes to hypoventilation, and whether circulating levels of leptin are involved in the pathogenesis of hypoventilation, which is often observed in OSAHS. We hypothesized that reduced levels of leptin may explain the increase of Paco 2 when BMI is similar in eucapnic and hypercapnic OSAHS patients. Materials and Methods The study population consisted of 185 male patients with OSAHS who were examined using polysomnography from April 2001 to December 2003. All patients were free from respiratory infection, heart failure, and other respiratory problems, including COPD, at the time of polysomnography. They were asked to complete a questionnaire on sleep symptoms, medical history, and medications. OSAHS was established on the basis of clinical and polysomnographic criteria. The average number of episodes of apnea and hypopnea per hour of sleep (the apnea-hypopnea index [AHI]) was calculated as the summary measurement of sleep-disordered breathing. In addition to clinical symptoms, an AHI of 5 was also used as a selection criterion. A male population with clinical symptoms of sleep apnea (n 520) was first divided into two groups according to AHI (AHI 5[n 426] and AHI 5[n 94]). Next, patients with AHI 5 were subclassified into two groups according to Paco 2 level (Paco 2 45 mm Hg [n 79] and Paco 2 45 mm Hg [n 327]). Hypercapnic OSAHS patients (Paco 2 45 mm Hg) were more obese and had a higher AHI and a lower arterial oxygen saturation (Sao 2 ) during sleep compared with eucapnic OSAHS patients. Then, eucapnic OSAHS patients (Paco 2 45 mm Hg) were further subclassified into two subgroups according to AHI (AHI 60 [n 45] and AHI 60 [n 302]). In addition, to compare hypercapnic and eucapnic patients matched for BMI and age, and to match the number of patients, those with an AHI 60 were further subclassified into two groups according to BMI (BMI 30 [n 61] and BMI 30 [n 241]). Finally, a subgroup with an AHI 60 (n 45) and a subgroup with an AHI 60 and a BMI 30 (n 61) were selected for the eucapnic group (Fig 1). Pulmonary function tests were performed to determine vital capacity (VC) and FEV 1 using a standard spirometer (Fudac-60; Fukuda Denshi; Tokyo, Japan). Arterial blood gas samples during room air breathing were drawn with the patient in the supine position and measured in a blood gas analyzer (Model 1312; Instrumental Laboratory; Milano, Italy). Overnight polysomnography (Compumedics; Melbourne, Australia) was performed between 9 pm and 6 am. Polysomnography consisted of continuous polygraphic recording from surface leads for EEG, electro-oculography, electromyography, ECG, thermistors for nasal and oral airflow, thoracic and abdominal impedance belts for respiratory effort, pulse oximetry for oxyhemoglobin level, tracheal microphone for snoring, and sensor for the position during sleep. Polysomnographic records were staged manually according to standard criteria. 12 Respiratory events were scored according to American Academy of Sleep Medicine criteria 13 : apnea was defined as complete cessation of airflow lasting 10 s, and hypopnea was defined as either a 50% reduction in airflow for 10 s or a 50% but discernible reduction in airflow accompanied either by a decrease in oxyhemoglobin saturation of 3% or arousal. Severity of OSAHS was determined based on the AHI and mean and lowest Sao 2. At 7 am on the morning after the sleep study, venous blood was obtained in the fasting state to measure leptin. Serum levels of leptin were determined by radioimmunoassay (Linco Research; St. Louis, MO) with intraassay and interassay coefficients of variation of 2.8 to 3.8% (n 10) and 0.4 to 4.6% (n 10), respectively. 14 Areas of SFA and VFA were measured by CT in a single cross-sectional scan at the level of the umbilicus. 15 The area of VFA was divided by that of SFA to calculate the VFA/SFA ratio. The study protocol was approved by the Research Ethics Committee of Chiba University School of Medicine, and all patients gave their informed consent prior to the study. Statistical Analysis The results are expressed as mean SEM. Age, BMI, pulmonary function parameters, and sleep parameters were compared between hypercapnic and eucapnic patients using the Mann- Whitney U test. Since data were not normally distributed, we used Spearman rank correlation coefficient to examine the association of two parameters. Analysis of covariance was used to compare the influence of BMI, VFA, and SFA on circulating leptin levels between hypercapnic and eucapnic patients. Logistic regression analysis was performed with Paco 2 as the dependent variable and leptin, BMI, VFA, SFA, mean Sao 2 during sleep, percentage of predicted VC, and percentage of predicted FEV 1 as explanatory variables; p 0.05 was considered statistically significant. 544 Clinical Investigations

Figure 1. Strategy for patient selection in this study. The hypercapnic group had Paco 2 levels 45 mm Hg (n 79). The eucapnic group included a subgroup with an AHI 60 (n 45) and a subgroup with an AHI 60 and a BMI 30 (n 61). PSG polysomnography. Results Baseline characteristics of the study population are shown in Table 1. None of the patients had an obstructive disease of the airway (FEV 1 /FVC 70%). Age, BMI, percentage of predicted VC, FEV 1 /FVC ratio, Pao 2, AHI, mean Sao 2 during sleep, and lowest Sao 2 during sleep were not significantly different between hypercapnic and eucapnic patients. The range of Paco 2 was 45.1 to 55.0 mm Hg, and the mean value was 46.6 mm Hg in the hypercapnic group; these figures were low compared to series of similar patients reported in the literature. 6,7 However, circulating leptin levels were significantly higher in the hypercapnic group compared with those in the eucapnic group (p 0.01). In the whole study group, circulating leptin levels correlated with BMI (r 0.56), VFA (r 0.24), SFA (r 0.47), and Paco 2 (r 0.24), but not with Pao 2, sleep mean Sao 2, or AHI. Hypercapnic patients had higher leptin levels than eucapnic patients when compared relative to BMI. The linear regression line between leptin levels and BMI in hypercapnic patients was shifted upward compared with that in eucapnic patients (p 0.05) [Fig 2]. When leptin values were plotted against VFA, they were distributed at a higher level in hypercapnic patients (p 0.05) [Fig 3]. Similarly, hypercapnic patients had higher leptin levels than eucapnic patients when compared relative to SFA (p 0.05) [Fig 4]. Logistic regression analysis with Paco 2 as the dependent variable showed that only circulating leptin was a predictor for the presence of Table 1 Baseline Characteristics of the Subjects* Characteristics Paco 2 45 mm Hg (n 106) Paco 2 45 mm Hg (n 79) p Value Age, yr 44.2 1.1 (22 72) 43.5 1.4 (22 72) NS BMI 32.5 0.3 (24.9 45.0) 33.2 0.8 (21.3 61.0) NS VC % predicted 96.5 1.6 (71 153) 84.9 0.6 (66 133) NS FEV 1 /FVC, % 84.2 0.6 (71 97) 85.4 1.7 (71 97) NS Pao 2,mmHg 80.6 1.0 (64.3 98.3) 77.9 1.1 (60.4 97.0) NS Paco 2,mmHg 40.9 0.3 (34.8 44.9) 46.6 0.4 (45.1 55.0) 0.01 AHI 52.9 2.2 (5.3 113.1) 47.4 2.8 (6.3 122.6) NS Mean Sao 2 during sleep, % 87.5 0.5 (72 96) 87.4 0.8 (70 97) NS Lowest Sao 2 during sleep, % 68.8 1.0 (39 87) 68.5 1.5 (43 91) NS Leptin, ng/ml 9.0 0.4 (1.5 26.1) 14.3 1.0 (2.9 47.5) 0.01 *Data are presented as mean SEM (range). NS not significant. www.chestjournal.org CHEST / 127 / 2/ FEBRUARY, 2005 545

Figure 2. Serum leptin levels vs percentage of BMI in patients with OSAHS. Each closed circle represents a hypercapnic patient, and each open circle represents a eucapnic patient. The dashed and solid regression lines describe the relationship between serum leptin levels and BMI in hypercapnic and eucapnic patients, respectively. Hypercapnic patients had higher leptin levels relative to BMI compared with eucapnic patients (p 0.05). hypercapnia ( 0.209, p 0.01), while BMI, VFA, SFA, mean Sao 2 during sleep, percentage of predicted VC, and FEV 1 /FVC ratio were not predictors. Discussion In the present study, we found that circulating levels of leptin were higher in hypercapnic patients with OSAHS than in eucapnic patients with OSAHS, although BMI, percentage of predicted VC, FEV 1 / FVC ratio, AHI, sleep mean Sao 2, VFA, and SFA were not significantly different between two groups. The levels of leptin relative to BMI, VFA, and SFA were higher in hypercapnic patients compared with those in eucapnic patients. Logistic regression analysis indicated that serum leptin was the only predic- Figure 3. Serum leptin levels vs VFA in patients with OSAHS. Each closed circle represents a hypercapnic patient, and each open circle represents a eucapnic patient. The dashed and solid regression lines describe the relationship between serum leptin levels and VFA in hypercapnic and eucapnic patients, respectively. Circulating leptin levels relative to VFA were higher in hypercapnic patients compared with those in eucapnic patients (p 0.05). 546 Clinical Investigations

Figure 4. Serum leptin levels vs SFA in patients with OSAHS. Each closed circle represents a hypercapnic patient, and each open circle represents a eucapnic patient. The dashed and solid regression lines describe the relationship between serum leptin levels and SFA in hypercapnic and eucapnic patients, respectively. Hypercapnic patients had higher leptin levels relative to SFA compared with eucapnic patients (p 0.05). tor for the presence of hypercapnia ( 0.21, p 0.01). These results suggest that leptin did not prevent hypoventilation in OSAHS, although leptin is a respiratory stimulant in mice. Phipps et al 16 reported that hyperleptinemia was associated with hypercapnic respiratory failure in 40 men and 16 women: eucapnia (n 44) and hypercapnia (n 12). Obesity is the major factor regulating circulating leptin, which is also influenced by gender and age. 17,18 We confirmed the results of Phipps et al 16 in a larger sample (n 185) of only men to avoid any gender effect on circulating levels of leptin. In addition, circulating levels of leptin were compared in hypercapnic and eucapnic patients with OSAHS matched for BMI and age. Moreover, whether the location of body fat deposits, ie, the distribution of VFA and SFA, contributes to hypoventilation was examined. VFA correlated weakly with levels of circulating leptin, compared with the relation between SFA and serum leptin, suggesting that the amount of visceral adiposity may not play a major role in the levels of circulating leptin. Circulating leptin concentrations were higher in obese subjects than in normal-weight subjects, although several factors other than the amount of body fat may contribute to the elevation of circulating leptin concentrations. 4,19,20 The mechanism of the increase in circulating leptin involves the induction of the ob gene. 17 Circulating leptin concentrations seem to be regulated by changes in body fat at the level of ob gene expression. 18 If leptin acts as it should, when adipocytes send the signal to the brain about the amount of adipose tissue, appetite will decrease and energy expenditure will increase, resulting in weight loss. Considering the fact that circulating leptin levels are elevated in most overweight individuals, obesity may be associated with leptin resistance. 21 23 In the present study, circulating leptin concentrations increased in parallel with BMI, although this relationship was not as constant as observed in inbred mice. 3,4 Obese C57BL/6J-Lep ob mice, which lack circulating leptin, exhibit respiratory depression and elevated Paco 2. Three days of leptin infusion restores ventilation, particularly during rapid eye movement sleep, in these obese mutant mice. Thus, leptin can prevent respiratory depression in obesity, while deficiency or reduced leptin levels may induce hypoventilation in some obese subjects. 9 Based on the findings of this mutant mice study, obese humans may acquire hypoventilation when circulating leptin levels are proportionately low. Therefore, we hypothesized that the reduced levels of leptin may explain the presence of hypoventilation in OSAHS. Alternatively, OSAHS patients may exhibit elevated Paco 2 when leptin levels in the CNS are relatively low, despite circulating leptin levels being high; ie, decreased central/circulating leptin ratio. 24,25 Obesity was suggested to be caused or related to the saturation of the leptin transport system from the periphery to the CNS. 26 Moreover, decreased sensitivity to leptin of the leptin-effector www.chestjournal.org CHEST / 127 / 2/ FEBRUARY, 2005 547

system in the CNS may also explain the presence of hypoventilation. This is analogous to what is observed in leptin-resistant mice, which bear a mutation in the leptin receptor gene (db/db mice). 27 Then, hypoventilation in OSAHS could be explained by two opposing hypotheses, insufficient brain leptin levels or impairment of the leptin transport system to the brain, and depressed sensitivity to leptin in the CNS. In our study, hypercapnic patients had higher leptin levels than eucapnic patients when compared relative to BMI; this may suggest that hypoventilation in OSAHS is partly due to depressed sensitivity to leptin in the CNS. However, human obesity is a complex disorder, and the pathophysiology of leptin is not as simple as it seems to be in rodent models of obesity. In addition, other mechanisms apart from fat mass could contribute to the increased leptin levels in OSAHS subjects. 19,20,28 In this study, hypercapnic patients were found to have higher leptin levels than eucapnic patients when compared relative to VFA and SFA. The linear regression line between leptin levels and VFA and SFA was shifted upward in hypercapnic patients compared with that in eucapnic patients (Fig 2, 3). It has been reported that leptin selectively decreases visceral adiposity in rats. 11 Whether leptin affects visceral adiposity was not clarified in this study. However, visceral adiposity was similar in hypercapnic and eucapnic patients, despite circulating leptin levels being higher in hypercapnic patients. The relation between cerebrospinal fluid (CSF) leptin and circulating leptin is best described by a logarithmic function. 24 The lower capacity of leptin transport from blood to brain in obese individuals may be one of the mechanisms for leptin resistance. 24 The higher levels of circulating leptin observed in hypercapnic patients in the present study may suggest a higher degree of resistance to leptin in these patients compared to eucapnic patients. However, leptin gene defects are rare in human obesity, 29 and there are no known functional or structural abnormalities of the brain leptin receptor in humans. We acknowledge one important limitation to our study: we did not obtain CSF samples from our patients to measure the levels of leptin. In addition, whether the ratio of CSF to blood leptin was modified or not under hypercapnia was unclear. Ideally, we should have performed the study in all subjects with and without hypercapnia matched for BMI. 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